Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Humans possess four TLS Pols that belong to the Y-family, Pols ?, ?, ?, and Rev1, and another Pol, Pol?, that belongs to the B-family. These TLS Pols employ highly specialized mechanisms for replicating through DNA lesions. Of these, Pol? is adept at extending opposite from bulky N2-dG minor groove lesions. Pol? can also carry out TLS opposite other types of DNA lesions; in that case, however, the lesion bypass ability is not limited to Pol?, as other TLS Pols can also function in their bypass. To elucidate the role of Pol? in lesion bypass in human cells, we will use a combined biochemical, genetic, and structural approach. In Aim 1, the role of two unique structural features of Pol?, (i) the N-clasp which allows Pol? to encircle DNA, and (ii) the openness of its active site towards the minor groove at the template-primer junction, will be analyzed by determining the effects of mutations on the extension reaction from N2-dG minor groove adducts and other types of DNA lesions as well. In Aim 2, the role of Pol? in promoting replication through different types of DNA lesions in human cells will be analyzed using a newly devised SV40-based plasmid system. Among the DNA lesions to be studied are 8- oxoguanine (8-oxoG) and thymine glycol (TG) that result from cellular oxidative DNA damage; the ring-opened N2-(3-hydroxyl propyl-2'-deoxygunaosine [(r)-3HOPdG] and the bulky trans-4-hydroxy-2-non-enal- deoxyguanosine (HNE-dG) adducts that result from the reaction of N2-dG with aldehydes or enals generated from free radical attack on lipids in membranes; and the multi-cyclic benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE) N2-dG adduct that results from exposure to environmental pollutants and carcinogens. To gain a deeper understanding of how Pol? actually performs TLS opposite these DNA lesions, in Aim 3, we will determine crystal structures of Pol? with 8-oxoG, TG, and cis-syn TT dimer, as well as with the N2-dG minor groove adducts of (r)-3HOPdG, HNE, and BPDE. The proficient ability of Pol? for extending from the C inserted opposite the N2-dG adducts by another DNA Pol, such as ? or Rev1, would ensure error-free replication through such minor groove DNA adducts. Since a large variety of N2-dG adducts are formed in human cells from cellular oxidative reactions and from exposure to chemical and environmental carcinogens, Pol? will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The proposed studies are highly relevant for cancer biology and etiology as they will reveal how human cells minimize the mutagenic and carcinogenic potential of DNA lesions.